1. Field of the Invention
The field of this invention concerns protein microspheres, such as are useful in biological treatments, experiments, and analyses, both in vivo and in vitro, and more particularly with remarkably versatile microspheres produced by a novel method.
The method and product are widely adaptable to use with numerous chemical and biochemical agents, and their derivatives, e.g. in carrying chemotherapeutic agents, alkaloids, halogenated compounds, hormones, lipids, nucleotides, porphyrins, steroids, vitamins, lectins, metals, their oxides, chlorides or sulfides, antibacterial and antifungal agents, enzymes, and like biologically useful agents, for example oxygen carrying molecules, into a host body for specific application at the point of need. The application is enabled selectively and controllably, without side effects resultant from excessive dosage or misdirection. A chemotherapeutic agent, for example, can be carried in a manner to bypass healthy cell areas, and therefore causing less side effects and at the same time enhancing therapeutic effectiveness. In certain cases, the agent may be released slowly and continuously, or may be released rapidly in the area to be treated. The biomodifying agents do not suffer any decrease in their effectiveness. The microspheres act to transport the various agents and may modify the kinetics of release of such agents.
The microspheres are both biocompatible and biodegradable, so that undesired foreign accumulations in the host body are avoided. Moreover, the method of microsphere production utilizes primarily ambient temperatures (although heating can be used if desired). The process does not require cooking of the albumin or any other destructive process. Through cooking or other heating, the incorporated agent may be damaged and suffer reduced effectiveness.
In particular, the invention relates to novel, improved protein microspheres of very small, very uniform particle size, and with great consistency and predictability, all with reagents and materials affording a clean, biocompatible and biodegradable microsphere product. The products of the method enable in vivo application of a wide variety of attached or incorporated chemical agents, without loss of their specific functions on a controlled, sustained release and/or selective, targeted basis, while the very small, uniform particle size enables passage through even capillaries to targeted areas. Being both biocompatible and biodegradable, the products of the method are powerful tools for pin-point implementation of therapies and treatments heretofore possible only theoretically or with undesired concomitant side effects.
2. Description of the Prior Art
Protein microspheres have been produced by the emulsification of an aqueous solution of a suitable protein, e.g. human serum albumin. See, Albumin microspheres as carrier of an inhibitor of leukocyte elastase: Potential therapeutic agent for emphysema, R. R. Martodam, et al., Proc. Nat'l Acad. Sci. U.S.A., Vol. 76, No. 5, pp. 2128-2132, May 1979. See also: Magnetically Responsive Microspheres and Other Carriers for the Biophysical Targeting of Antitumor Agents, K. J. Widder et al., Advances in Pharmacology and Chemotherapy, Vol. 16, pp. 213-271, especially pages 233-239.
As will be apparent from a consideration of the latter of the foregoing articles, albumin microspheres have been prepared by emulsifying water solutions of human serum albumin in cottonseed oil, followed by stabilization with heat which is highly disadvantageous in at least two respects: (1) the albumin becomes highly denatured and thus is recognized by the host body as foreign and is therefore rapidly eliminated; and (2) where heat sensitive chemicals are used, the agent is destroyed during the heating process. In addition, the process requires the subsequent extraction of the oil by ether washing, and sieving to classify as to size.
The concept of forming microspheres is also disclosed and claimed in U.S. Pat. No. 4,107,288 issued on Aug. 15, 1978 to Oppenheim et al. for "Injectable Compositions, Nanoparticles Useful Therein, And Process Of Manufacturing Same" ("Oppenheim"). However, the process disclosed and claimed in Oppenheim produces an aggregation of particles and further requires the process to be carried out at elevated temperatures which can denature the albumin and/or destroy heat sensitive chemicals. The problem with the Oppenheim process is that proceeding from the dissolving step directly to the step where the desolvating agent is added produces an uncontrolled reaction where the particles lump together too fast and an aggregation occurs. This is apparent from Column 3, lines 40 to 50 of the Oppenheim patent. If the aggregation formed is too clumpy, then alcohol must be added to reverse the desolvation. The Oppenheim method may be satisfactory with gelatin compounds but with albumin which is not as thick and viscous as gelatin the result with the Oppenheim process is uncontrolled aggregation when the dissolving step is immediately followed by addition of the desolvating agent.
A suitable surfactant must be employed to result in monodispersed microspheres with less than 0.1% aggregates. The critical difference between this invention and that of Oppenheim is the discovery that a suitable surfactant or detergent must be used to prepare the surface of the protein cross-linking agent complex for microsphere formation. Oppenheim teaches that the nature of the surfactant or suspending agent is not critical except that the surfactant or suspending agent should remain in solution throughout the process (column 4, line 25 to 27). The use of surfactant is not mentioned in his examples 1, 2, 3, 4, 5, 6, 7 or 8. The products were comprised of aggregated spherical particles (column 6, line 5). It is not clear if each sphere is about 330-660 nanometers in diameter, while the entire aggregate can be millimeter (thousands of microns in size) in diameter as I have found. Oppenheim's example 9 mentions "a suitable concentration of surfactant: 0.5 to 3.0% w/v (column 6, line 38). However neither the identity of the surfactant nor its importance is identified. Therefore, Oppenheim teaches away from the importance of the surfactant.
Accordingly, a significant need exists for a process by which microspheres can be created in a controlled reaction to thereby avoid aggregation and further permit uniform homogeneous microspheres to be formed which can be used with albumin and other compounds which are not as viscous as gelatin.
In addition, there is also a need for microsphere preparations with controlled porosity of the microspheres. In certain conditions, biological molecules to be deployed to the host would require protection from destructive mechanisms of the host. Protection can be afforded by a porous matrix of the microsphere, with the biological molecule, such as hemoglobin, to be covalently bonded to the interior of the microsphere. This requires that the relatively large biological molecule be able to penetrate the surface of the microspheres, which is dependent on the sizes of the pores on the surface and interior of the microspheres. One theoretical approach to incorporate such molecules in the interior of the microspheres would be to link the biological molecule to the protein molecules before the formation of microspheres. Such an approach would presume a random distribution of the biological molecule such that a majority of the biological molecules will result in the interior of the microspheres. Such may not always be the case if a biological molecule is highly hydrophilic, or is so bulky as to interfere with the formation of microspheres which in the absence of such biological molecules may be easily and controllably assembled. More importantly, the conditions used to produce microspheres may be detrimental to the biological stability and therefore to the function of the biological molecule. For example, the use of a high concentration of ammonium sulfate solution in Oppenheim's approach will certainly destroy the hemoglobin portion of any pre-formed hemoglobin-gelatin complex.
The primary embodiment of my methodology allows a low concentration of cross-linking agent to lightly cross-link protein molecules before their desolvation in the presence of a suitable surfactant. The sizes of the microspheres as well as the porosity of the microspheres formed is large when the concentration of cross-linking agent used is low. This is due to a lower number of internal bonds formed under well defined and easily controllable conditions.
A modification of Oppenheim's approach may also be theoretically entertained, namely that cross-linking agents may be added to the gelatin in their example before the step of "desolvation region", thus achieving a procedure similar to my invention. However, Oppenheim clearly indicates that the desolvation region can easily be overshot which will lead to uncontrolled clumping and which requires the addition of a reversal agent. If Oppenheim attempted pre-linkage of gelatin molecules, once the desolvation region is overshot, the cross-linkage of the gelatin in the presence of the cross-linking agent will prevent reversal of the condition, resulting in a highly clumped useless preparation. Furthermore, Oppenheim teaches away from the importance of a suitable surfactant, which mainly accounts for the labile condition of the "desolvation region."
My approach allows the use of a range of low concentration of cross-linking material; easily controlled duration of interaction between the cross-linking agent and the protein molecules to achieve partial and light initial cross-linkage; stability of such protein cross-linkage agent complexes; and more importantly, stabilization of microspheres by the presence of a suitable surfactant before the addition of the desolvating agent. Thus we control the sizes of the microspheres, the distribution of sizes of microspheres, and the porosity of the resultant microspheres, all of which properties are important to the specific task of carrying certain relatively large and easily biodegradable biological molecules.
In another embodiment of my process, I formed reversible microspheres by addition of a desolvation agent to unlinked protein molecules in the presence of a suitable surfactant. Subsequently, a low concentration of cross-linking agent is used to gently and irreversibly form internal bonding within the microspheres. Although superficially this embodiment may appear to resemble that of Oppenheim's approach, my method will result in well controlled microsphere sizes as well as porosity, again by virtue of the absence of an unstable "desolvation region" with the associated disadvantages discussed above.